The Heparanase (HPA) BioAssay(TM) ELISA Kit is a quantitative sandwich assay for the detection of HPA in human serum, plasma and other biological fluids. Detection Detection Range: 0.16-10ng/ml Sensitivity: 0.1ng/ml Precision: Intra-Assay %CV: <10% Inter-Assay %CV: <10% Specificity: Recognizes human HPA. No significant crossreactivity or interference between human HPA and analogues was observed. Test Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to HPA. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to HPA. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Free components are washed away. The substrate solution is added, only those wells that contain HPA, biotin-conjugated antibody and enzyme-conjugated Avidin will apear blue in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450nm 2nm. The OD value is proportional to the concentration of human HPA. The concentration of HPA in the sample is then determined by comparing the O.D. of the sample to the standard curve. Kit Components: *383283A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use *383283B: Standard, 2x1vial 383283C: Standard Diluent, 1x20ml *383283D: Biotin Detection, 100X, 1x120ul *383283E: HRP Conjugate, 100X, 1x120ul 383283F: Biotin Detection Diluent, 1x14ml 383283G: HRP Diluent, 1x14ml 383283H: Substrate, 1x10ml 383283K: Stop Solution, 1x10ml 383283L: Wash Buffer, 25X, 1x30ml Storage and Stability: Store *383283A, *383283B, *383283D and *383283E at -20C. Store all the other components at 4C. Unused kit is stable for 6 months after receipt. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap. Assay Procedure Summary: 1. Prepare all reagents, samples and standards. 2. Add 100ul standard or sample to each well. Incubate 90 minutes at 37C. 3. Aspirate and add 100ul prepared Biotin Working Solution. Incubate 1 hour at 37C. 4. Aspirate and wash 3 times. 5. Add 100ul prepared HRP Detection Working Solution. Incubate 30 minutes at 37C. 6. Aspirate and wash 5 times. 7. Add 90ul Substrate. Incubate 15 minutes at 37C. 8. Add 50ul Stop Solution. Read at 450nm immediately.
